Enhanced oxidative stress, damage and inadequate antioxidant defense contributes towards insufficient recovery in water deficit stress and heat stress combination compared to either stresses alone in Chenopodium album (Bathua).

Chenopodium album (common name Bathua) is a widely adapted weed plant facing wide array of temperatures (5-45 °C) during growth and development in North India. Antioxidant defense was studied in C. album leaves under water deficit stress, heat stress, water deficit stress and heat stress combination and water deficit preconditioning followed by heat stress. C. album plants subjected to water deficit stress and heat stress combination showed higher decline in water relations and lesser recovery compared to either stresses alone. Highest H2O2 content, lipid peroxidation and protein damage were observed in plants experiencing water deficit stress and heat stress combination which was coupled with less induction in activities of SOD, CAT and all AsA-GSH cycles enzymes and decline in AsA and GSH pool compared to plants subjected to either stress alone. Water deficit preconditioned C. album plants maintained higher activities of antioxidant defense enzymes and metabolites such as SOD, CAT, POX, DHAR, GSH content and AsA/DHA and GSH/GSSG ratios compared to non-preconditioned plants under heat stress. This is the first holistic report on effect of water deficit stress and heat stress combination and water deficit preconditioning followed by heat stress on ROS, damage and antioxidant defense including enzymes and metabolites in C. album. Water deficit stress and heat stress combination was more detrimental in C. album than either of the stresses alone as decline in water relations and increase in oxidative stress and damage was coupled with a decline in antioxidant defense both in enzymes i.e. SOD, CAT and AsA-GSH cycles enzymes and metabolites i.e. AsA and GSH content. Water deficit preconditioning followed by recovery resulted in induction of co-ordinated antioxidant defense in terms of both enzyme activities and metabolites during subsequent heat stress in C. album. Enhanced CAT activity and higher redox pool played a major role in cross tolerance in water deficit preconditioned C. album plants under heat stress.

Reproductive sink enhanced drought induced senescence in wheat fertile line is associated with loss of antioxidant competence compared to its CMS line.

Reproductive sinks regulate monocarpic senescence in wheat as desinking delayed flag leaf senescence under irrigated condition. In this study, wheat cv. HW 2041 and its isonuclear male sterile line (CMS) were subjected to post-anthesis water deficit stress to understand the association between sink strength, senescence and drought response in relation to oxidative stress and antioxidant defense at cellular and sub-cellular level. CMS plants maintained better water relations and exhibited delayed onset and progression of flag leaf senescence in terms of green leaf area, chlorophyll and protein content than fertile plants under water deficit stress (WDS). Delayed senescence in CMS plants under water deficit stress was associated with less reactive oxygen species generation, lower damage to membranes and better antioxidant defense both in terms of antioxidant enzyme activities and metabolite content compared to fertile plants. Expression of some senescence associated genes (SAGs) such as WRKY transcription factor (WRKY53), glutamine synthetase1 (GS1), wheat cysteine protease (WCP2) and wheat serine protease (WSP) was lower while catalse 2 (CAT2) transcript levels were higher in the CMS plants compared to HW2041 during senescence under water deficit stress. Antioxidant defense in chloroplasts was better in CMS line under water deficit stress compared to HW2041. This is the first report showing that reproductive sink enhanced drought induced senescence in flag leaf of wheat fertile line is associated with higher oxidative stress and damage and loss of antioxidant competence compared to its sterile line under water deficit stress. Higher expression of some SAGs and decline in superoxide dismutase and ascorbate peroxidase activity in the chloroplasts also contributed to the accelerated senescence in fertile line compared to its CMS line under WDS.

Wheat cultivars differing in heat tolerance show a differential response to oxidative stress during monocarpic senescence under high temperature stress.

Wheat crop may experience heat stress during post-anthesis phase associated with oxidative stress, enhanced senescence, and reduced productivity. Stay green is a desirable character for the selection for heat tolerance in wheat. In the present study, antioxidant metabolism was studied under post-anthesis heat stress in field during monocarpic senescence by comparing two wheat genotypes, namely Hindi62 (heat tolerant and delayed senescent) and PBW343 (heat susceptible and early senescent). Hindi62 exhibited lesser oxidative stress, membrane damage, and coordinated antioxidant defense as compared to PBW343 under heat stress during post-anthesis stage. Higher activity of SOD, CAT, APX, GR, and MDHAR under heat stress contributed towards delayed senescence in Hindi62 compared to PBW343. GSH/GSSG ratio was also maintained at higher level in Hindi62 under heat stress compared to PBW343 during senescence. Hence, the present study clearly shows that upregulated level of the total antioxidant capacity during grain development contributed towards delayed senescence and heat tolerance in Hindi62 compared to the heat-susceptible PBW343.

Delayed expression of SAGs correlates with longevity in CMS wheat plants compared to its fertile plants.

Reproductive sinks regulate monocarpic senescence in crop plants. Monocarpic senescence was studied in wheat fertile (cv. HW 2041) and its isonuclear cytoplasmic male sterile (CMS) line. CMS plants exhibited slower rate of senescence accompanied by longer green leaf area duration and slower deceleration in chlorophyll, protein content, PN and rubisco content coupled with lower protease activities than fertile (F) plants. CMS plants also exhibited lower ROS levels and less membrane damage than F plants. CMS plants maintained better antioxidant defense, less oxidative damage in chloroplast and higher transcript levels of both rbcL and rbcS genes during senescence than F plants. F plants exhibited early induction and higher expression of SAGs like serine and cysteine proteases, glutamine synthetases GS1 and GS2, WRKY53 transcription factor and decline in transcript levels of CAT1 and CAT2 genes than CMS plants. Hence, using genetically fertile and its CMS line of wheat it is confirmed that delayed senescence in the absence of reproductive sinks is linked with slower protein oxidation, rubisco degradation and delayed activation of SAGs. Better antioxidant defense in chloroplasts at later stages of senescence was able to mitigate the deleterious effects of ROS in CMS plants. We propose that delayed increase in ROS in cytoplasmic male sterile wheat plants resulted in delayed activation of WRKY53, SAGs and the associated biochemical changes than fertile plants.

Genomic associations for drought tolerance on the short arm of wheat chromosome 4B.

Drought is a major constraint to maintaining yield stability of wheat in rain fed and limited irrigation agro-ecosystems. Genetic improvement for drought tolerance in wheat has been difficult due to quantitative nature of the trait involving multiple genes with variable effects and lack of effective selection strategies employing molecular markers. Here, a framework molecular linkage map was constructed using 173 DNA markers randomly distributed over the 21 wheat chromosomes. Grain yield and other drought-responsive shoot and root traits were phenotyped for 2 years under drought stress and well-watered conditions on a mapping population of recombinant inbred lines (RILs) derived from a cross between drought-sensitive semidwarf variety "WL711" and drought-tolerant traditional variety "C306". Thirty-seven genomics region were identified for 10 drought-related traits at 18 different chromosomal locations but most of these showed small inconsistent effects. A consistent genomic region associated with drought susceptibility index (qDSI.4B.1) was mapped on the short arm of chromosome 4B, which also controlled grain yield per plant, harvest index, and root biomass under drought. Transcriptome profiling of the parents and two RIL bulks with extreme phenotypes revealed five genes underlying this genomic region that were differentially expressed between the parents as well as the two RIL bulks, suggesting that they are likely candidates for drought tolerance. Syntenic genomic regions of barley, rice, sorghum, and maize genomes were identified that also harbor genes for drought tolerance. Markers tightly linked to this genomic region in combination with other important regions on group 7 chromosomes may be used in marker-assisted breeding for drought tolerance in wheat.

Leaf senescence and abiotic stresses share reactive oxygen species-mediated chloroplast degradation.

Leaf senescence is a genetically programmed decline in various cellular processes including photosynthesis and involves the hydrolysis of macromolecules such as proteins, lipids, etc. It is governed by the developmental age and is induced or enhanced by environmental stresses such as drought, heat, salinity and others. Internal factors such as reproductive structures also influence the rate of leaf senescence. Reactive oxygen species (ROS) generation is one of the earliest responses of plant cells under abiotic stresses and senescence. Chloroplasts are the main targets of ROS-linked damage during various environmental stresses and natural senescence as ROS detoxification systems decline with age. Plants adapt to environmental stresses through the process of acclimation, which involves less ROS production coupled with an efficient antioxidant defence. Chloroplasts are a major site of protein degradation, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is rapidly and selectively degraded during senescence and stress. The process of protein degradation is initiated by ROS and involves the action of proteolytic enzymes such as cysteine and serine proteases. The mechanism of Rubisco degradation still remains to be elucidated. The molecular understanding of leaf senescence was achieved through the characterization of senescence-associated genes and various senescence mutants of Arabidopsis, which is a suitable model plant showing monocarpic senescence. The regulation of senescence involves many regulatory elements composed of positive and negative elements to fine-tune the initiation and progression of senescence. This review gives an overview on chloroplast protein degradation during leaf senescence and abiotic stresses and also highlights the role of ROS management in both processes.

Chloroplasts and mitochondria have multiple heat tolerant isozymes of SOD and APX in leaf and inflorescence in Chenopodium album.

Thermal stability of antioxidant defense enzymes superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate peroxidase (APX, EC 1.11.1.11) was studied in chloroplasts and mitochondria of leaf and inflorescence in heat adaptive weed Chenopodium album. Leaf samples were taken in March (31°C/14°C) and young inflorescence (INF) was sampled at flowering in April (40°C/21°C). Leaf and INF chloroplast and mitochondrial fractions were subjected to elevated temperatures in vitro (5-100°C) for 30'. SOD and APX showed activity even after boiling treatment in both chloroplast and mitochondria of leaf and INF. SOD was more heat stable than APX in both chloroplasts and mitochondria in both the tissues. Chloroplast contained more heat stable SOD and APX isozymes than mitochondria in both leaf and INF. To the best of our knowledge this is the first report showing presence of thermostable APX isozymes (100°C for 30') in chloroplasts and mitochondria in C. album. Heat stable isozymes of SOD and APX in chloroplasts and mitochondria in leaves and inflorescence may contribute to heat tolerance in C. album.

Superoxide dismutase and ascorbate peroxidase are constitutively more thermotolerant than other antioxidant enzymes in Chenopodium album.

Thermal stability of antioxidant defense enzymes was investigated in leaf and inflorescence of heat adaptive weed Chenopodium album. Leaf samples were taken at early and late seedling stage in December (LD, 20 °C/4 °C) and March (LM, 31 °C/14 °C). Young inflorescence (INF) was sampled at flowering in April (40 °C/21 °C). LD, LM and INF crude protein extracts were subjected to elevated temperatures (5 to 100 °C) for 30'. Superoxide dismutase (SOD) was the most heat stable enzyme followed by Ascorbate peroxidase (APX). Two heat stable SOD isozymes were visible on native-PAGE at 100 °C in both leaf and INF. Some heat stable APX isozymes were more abundant in INF than leaf. Thermostability of catalase (CAT) increased with age and increasing ambient temperatures in leaves. CAT activity was observed up to 60 °C in leaves and INF while peroxidase (POX) retained activity up to 100 °C in INF due to one thermostable isozyme. Glutathione reductase (GR), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and monodehydroascorbate reductase (MDHAR) showed activity up to 70 °C in both leaves and INF. DHAR activity was stable up to 60 °C while GR and MDHAR declined sharply after 40 °C. Constitutive heat stable isozymes of SOD and APX in leaves and INF may contribute towards heat tolerance in C. album.

Antioxidant response of wheat roots to drought acclimation.

Wheat (Triticum aestivum L.) seedlings of a drought-resistant cv. C306 were subjected to severe water deficit directly or through stress cycles of increasing intensity with intermittent recovery periods. The antioxidant defense in terms of redox metabolites and enzymes in root cells and mitochondria was examined in relation to membrane damage. Acclimated seedlings exhibited higher relative water content and were able to limit the accumulation of H(2)O(2) and membrane damage during subsequent severe water stress conditions. This was due to systematic up-regulation of superoxide dismutase, ascorbate peroxidase (APX), catalase, peroxidases, and ascorbate-glutathione cycle components at both the whole cell level as well as in mitochondria. In contrast, direct exposure of severe water stress to non-acclimated seedlings caused greater water loss, excessive accumulation of H(2)O(2) followed by elevated lipid peroxidation due to the poor antioxidant enzyme response particularly of APX, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, and ascorbate-glutathione redox balance. Mitochondrial antioxidant defense was found to be better than the cellular defense in non-acclimated roots. Termination of stress followed by rewatering leads to a rapid enhancement in all the antioxidant defense components in non-acclimated roots, which suggested that the excess levels of H(2)O(2) during severe water stress conditions might have inhibited or down-regulated the antioxidant enzymes. Hence, drought acclimation conferred enhanced tolerance toward oxidative stress in the root tissue of wheat seedlings due to both reactive oxygen species restriction and well-coordinated induction of antioxidant defense.

Delayed wheat flag leaf senescence due to removal of spikelets is associated with increased activities of leaf antioxidant enzymes, reduced glutathione/oxidized glutathione ratio and oxidative damage to mitochondrial proteins.

Removal of reproductive 'sink' i.e. spikelets from wheat at anthesis delays the rate of flag leaf senescence. In this work, the antioxidant defense was studied in the flag leaf of Triticum aestivum cv. Kalyansona plants showing normal (S + plants) and delayed senescence via removal of spikelets (S- plants). This was done by measurement of metabolites and activities of enzymes such as superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase. S- plants had higher reduced glutathione/oxidized glutathione (GSH/GSSG) ratio and antioxidant enzyme activities than the control plants and the differences were apparent from 21 days after anthesis (DAA). The removal of the reproductive sink led to an increased antioxidant defense which may be contributing towards the delayed flag leaf senescence in wheat. Chloroplasts and mitochondria, important sources of ROS, were isolated at two stages representing early (7 DAA) and late (21 DAA) senescence. Oxidative damage to proteins was studied in these organelles in relation to SOD and APX. Mitochondria had higher levels of damaged proteins than chloroplasts at 7 DAA in both S+ and S- plants. Higher damage was related to the lower antioxidant enzyme levels of SOD and APX in mitochondria as compared to chloroplasts.

Purification and characterization of thermostable monomeric chloroplastic Cu/Zn superoxide dismutase from Chenopodium murale.

Superoxide dismutase is the first line of defense against oxidative stress and thus helps in maintaining the cellular integrity. Chenopodium murale, a weed species adapted to widely varying climatic conditions faces extremes of temperatures ranging from 4 °C to 45 °C (Tmax) during growth and development. From this plant, we have purified a thermostable chloroplastic Cu/Zn superoxide dismutase (Chl Cu/Zn SOD) to homogeneity using minimal steps. Incubation of lysed chloroplasts at 70 °C for 1h reduced the interference of cytosolic SOD isoforms and reduced the protein content by 75 %. Chloroplastic SOD was purified from the heat stable fraction by gel filtration chromatography. The purified enzyme had a native molecular weight of 24 kDa, a half-life of 47.9 min at 80 °C and showed a single band at 24 kDa on SDS-PAGE. The N-terminus contained the conserved amino acids of chl Cu-Zn SOD. The Chl Cu/Zn SOD protein and its activity were enhanced under very high temperatures, high light intensities and in water stress/recovered C. murale plants under controlled environment conditions. Chl Cu/Zn SOD was also one of the predominant isoforms throughout growing period in field grown plants and declined during senescence. The Chl Cu/Zn SOD activity increased with the increase in ambient temperature and peaked in April with a 45 °C Tmax. These results clearly indicate that the chloroplastic Cu/Zn SOD is stably expressed at extreme environmental conditions. The presence of stable monomeric chloroplastic Cu/Zn SOD might help the plants to maintain the cellular homeostatis against adverse environmental conditions.

The developing reproductive 'sink' induces oxidative stress to mediate nitrogen mobilization during monocarpic senescence in wheat.

Removal of reproductive 'sink,' i.e., spikelets from wheat, after anthesis delays the rate of flag leaf senescence. Oxidative stress and the oxidative damage to proteins were studied in relation to nitrogen mobilization in wheat plants showing normal and delayed senescence. Wheat plants lacking a reproductive sink showed decreased oxidative stress, lower lipid peroxidation and maintained higher protein, oxidatively damaged proteins, and nitrogen levels as compared to plants with reproductive sink during monocarpic senescence. Oxidative damage to the proteins when not followed by high proteolytic activities led to a slower nitrogen mobilization in wheat plants lacking a reproductive sink. Thus, the influence of the reproductive sink was due to its ability to drive forward the nitrogen mobilization process through high ROS levels which mediated both damage to the proteins and influenced proteolytic activities.

Heat-stable chloroplastic Cu/Zn superoxide dismutase in Chenopodium murale.

Chenopodium murale is a weed species having wide adaptation to different climatic regimes and experiences a temperature range of 5-45 degrees C during its life span. Higher temperatures may result in heat stress, which induces higher ROS production leading to oxidative stress in the plant. Superoxide dismutase enzyme (SOD, EC.1.15.1.1) is ubiquitous, being widely distributed among O(2)(-) consuming organisms and is the first line of defense against oxidative stress. In this study, we have characterized the thermostability of the SOD isozymes from C. murale in vitro. The leaf protein extracts, thylakoidal and stromal fractions were subjected to elevated temperatures ranging from 50 degrees C to boiling and analyzed for activity and isoform pattern of SOD. Out of six SOD isoforms, SOD V showed stability even after boiling the extract for 10min. Under high temperature treatment (>60 degrees C) there was an appearance of a new SOD band with higher electrophoretic mobility. The inhibitor studies and subcellular analysis revealed that the SOD V isoform was a chloroplastic Cu/Zn SOD. The stromal Cu/Zn SOD (SOD V) was more stable than the co-migrating thylakoidal isozyme at 80 degrees C and boiling for 10min. Hence, we report an unusual, constitutive thermostable chloroplastic Cu/Zn SOD from C. murale, which may contribute towards its heat tolerance.

Drought acclimation reduces O2*- accumulation and lipid peroxidation in wheat seedlings.

Abiotic stresses cause ROS accumulation, which is detrimental to plant growth. It is well known that acclimation of plants under mild or sub-lethal stress condition leads to development of resistance in plants to severe or lethal stress condition. The generation of ROS and subsequent oxidative damage during drought stress is well documented in the crop plants. However, the effect of drought acclimation treatment on ROS accumulation and lipid peroxidation has not been examined so far. In this study, the effect of water stress acclimation treatment on superoxide radical (O(2)(-z.rad;)) accumulation and membrane lipid peroxidation was studied in leaves and roots of wheat (Triticum aestivum) cv. C306. EPR quantification of superoxide radicals revealed that drought acclimation treatment led to 2-fold increase in superoxide radical accumulation in leaf and roots with no apparent membrane damage. However under subsequent severe water stress condition, the leaf and roots of non-acclimated plants accumulated significantly higher amount of superoxide radicals and showed higher membrane damage than that of acclimated plants. Thus, acclimation-induced restriction of superoxide radical accumulation is one of the cellular processes that confers enhanced water stress tolerance to the acclimated wheat seedlings.

Differential response of antioxidant enzymes in leaves of necrotic wheat hybrids and their parents.

The leaves of necrotic hybrid of wheat (Triticum aestivum L.) exhibited high superoxide content associated with increased lipid peroxidation and membrane damage in earlier studies (Khanna-Chopra et al. 1998, Biochem Biophys Res Commun 248: 712-715; Dalal and Khanna-Chopra 1999, Biochem Biophys Res Commun 262: 109-112). In the present study, we investigated the activities of the antioxidant enzymes in the leaves of necrotic wheat hybrids, KalyansonaxC306 (KxC) and WL711xC306 (WLxC) and their parents at different developmental stages. The KxC hybrid exhibited more severe necrosis than WLxC. In KxC, superoxide dismutase (SOD) activity showed no increase over the parents, while WLxC showed an early increase, but it was possibly insufficient to scavenge increased superoxide. Activities of guaiacol peroxidase, ascorbate peroxidase and glutathione reductase were enhanced, while catalase exhibited a decrease in activity, with the appearance of visible necrosis in both the hybrids. The isozyme profile of the antioxidant enzymes was similar in the hybrids and their parents. One existing isoform of guaiacol peroxidase showed an early appearance in the hybrid and increased in intensity with the progression of necrosis. The results reveal a differential response of antioxidant enzymes in necrotic wheat hybrids as compared to their parents. The response differed in magnitude at developmental stages of the leaves, which might be related to the intensity of necrosis expressed by the hybrids.